Glucagon-like peptide-1 receptor (GLP-1R) activation promotes insulin secretion from pancreatic beta cells, causes weight loss, and is an important pharmacological target in type 2 diabetes (T2D). ...Like other G protein-coupled receptors, the GLP-1R undergoes agonist-mediated endocytosis, but the functional and therapeutic consequences of modulating GLP-1R endocytic trafficking have not been clearly defined. Here, we investigate a series of biased GLP-1R agonists with variable propensities for GLP-1R internalization and recycling. Compared to a panel of FDA-approved GLP-1 mimetics, compounds that retain GLP-1R at the plasma membrane produce greater long-term insulin release, which is dependent on a reduction in β-arrestin recruitment and faster agonist dissociation rates. Such molecules elicit glycemic benefits in mice without concomitant increases in signs of nausea, a common side effect of GLP-1 therapies. Our study identifies a set of agents with specific GLP-1R trafficking profiles and the potential for greater efficacy and tolerability as T2D treatments.
Extracellular vesicles (EVs) are released by shedding during different physiological processes and are increasingly thought to be new potential biomarkers. However, the impact of pre-analytical ...processing phases on the final measurement is not predictable and for this reason, the translation of basic research into clinical practice has been precluded. Here we have optimized a simple procedure in combination with polychromatic flow cytometry (PFC), to identify, classify, enumerate, and separate circulating EVs from different cell origins. This protocol takes advantage of a lipophilic cationic dye (LCD) able to probe EVs. Moreover, the application of the newly optimized PFC protocol here described allowed the obtainment of repeatable EVs counts. The translation of this PFC protocol to fluorescence-activated cell sorting allowed us to separate EVs from fresh peripheral blood samples. Sorted EVs preparations resulted particularly suitable for proteomic analyses, which we applied to study their protein cargo. Here we show that LCD staining allowed PFC detection and sorting of EVs from fresh body fluids, avoiding pre-analytical steps of enrichment that could impact final results. Therefore, LCD staining is an essential step towards the assessment of EVs clinical significance.
Tissue-specific transcriptional regulation is central to human disease. To identify regulatory DNA active in human pancreatic islets, we profiled chromatin by formaldehyde-assisted isolation of ...regulatory elements coupled with high-throughput sequencing (FAIRE-seq). We identified ∼80,000 open chromatin sites. Comparison of FAIRE-seq data from islets to that from five non-islet cell lines revealed ∼3,300 physically linked clusters of islet-selective open chromatin sites, which typically encompassed single genes that have islet-specific expression. We mapped sequence variants to open chromatin sites and found that rs7903146, a TCF7L2 intronic variant strongly associated with type 2 diabetes, is located in islet-selective open chromatin. We found that human islet samples heterozygous for rs7903146 showed allelic imbalance in islet FAIRE signals and that the variant alters enhancer activity, indicating that genetic variation at this locus acts in cis with local chromatin and regulatory changes. These findings illuminate the tissue-specific organization of cis-regulatory elements and show that FAIRE-seq can guide the identification of regulatory variants underlying disease susceptibility.
Diseases associated with Xylella fastidiosa have been described mostly in North and South America. However, during the last five years, widespread X. fastidiosa infections have been reported in a ...constrained area of the Apulia region (southern Italy), in olives trees suffering a severe disease, denoted as Olive Quick Decline Syndrome (OQDS). Because many xylem sap-feeding insects can function as vectors for the transmission of this exotic pathogen in EU, several research programs are ongoing to assess the role of candidate vectors in the spread of the infections. Initial investigations identified Philaenus spumarius (L.) as the predominant vector species in the olive orchards affected by the OQDS. Additional experiments have been carried out during 2016 and 2017 to assess the role of other species. More specifically, adults of the spittlebugs Philaenus italosignus Drosopolous and Remane, Neophilaenus campestris (Fallen) and of the planthopper Latilica tunetana (Matsumura) (Issidae) have been tested in transmission experiments to assess their ability to acquire the bacterium from infected olives and to infect different susceptible hosts (olives, almond, myrtle –leaf milkwort, periwinkle). Acquisition rates determined by testing individual insects in quantitative PCR assays, ranging from 5.6% in N. campestris to 22.2% in P. italosignus, whereas no acquisition was recorded for L. tunetana. Successful transmissions were detected in the recipient plants exposed to P. italosignus and N. campestris, whereas no trasmissions occurred with L. tunetana. The known vector Philaenus spumarius has been included in all the experiments for validation. The systematic surveys conducted in 2016 and 2017 provided further evidence on the population dynamics and seasonal abundance of the spittlebug populations in the olive groves.
Philaenus spumarius is a cosmopolitan species that has become a major threat to European agriculture being recognized as the main vector of the introduced plant pathogen Xylella fastidiosa, the agent ...of the “olive quick decline syndrome”, a disease which is devastating olive orchards in southern Italy. Wolbachia are bacterial symbionts of many insects, frequently as reproductive parasites, sometime by establishing mutualistic relationships, able to spread within host populations. Philaenus spumarius harbors Wolbachia, but the role played by this symbiont is unknown and data on the infection prevalence within host populations are limited. Here, the Wolbachia infection rate was analyzed in relation to the geographic distribution and the genetic diversity of the Italian populations of P. spumarius. Analysis of the COI gene sequences revealed a geographically structured distribution of the three main mitochondrial lineages of P. spumarius. Wolbachia was detected in half of the populations sampled in northern Italy where most individuals belonged to the western-Mediterranean lineage. All populations sampled in southern and central Italy, where the individuals of the eastern-Mediterranean lineage were largely prevalent, were uninfected. Individuals of the north-eastern lineage were found only in populations from the Alps in the northernmost part of Italy, at high altitudes. In this area, Wolbachia infection reached the highest prevalence, with no difference between north-eastern and western-Mediterranean lineage. Analysis of molecular diversity of COI sequences suggested no significant effect of Wolbachia on population genetics of P. spumarius. Using the MLST approach, six new Wolbachia sequence types were identified. Using FISH, Wolbachia were observed within the host’s reproductive tissues and salivary glands. Results obtained led us to discuss the role of Wolbachia in P. spumarius, the factors influencing the geographic distribution of the infection, and the exploitation of Wolbachia for the control of the vector insect to reduce the spread of X. fastidiosa.
Phytoplasmas, uncultivable phloem-limited phytopathogenic wall-less bacteria, represent a major threat to agriculture worldwide. They are transmitted in a persistent, propagative manner by ...phloem-sucking Hemipteran insects. Phytoplasma membrane proteins are in direct contact with hosts and are presumably involved in determining vector specificity. Such a role has been proposed for phytoplasma transmembrane proteins encoded by circular extrachromosomal elements, at least one of which is a plasmid. Little is known about the interactions between major phytoplasma antigenic membrane protein (Amp) and insect vector proteins. The aims of our work were to identify vector proteins interacting with Amp and to investigate their role in transmission specificity. In controlled transmission experiments, four Hemipteran species were identified as vectors of "Candidatus Phytoplasma asteris", the chrysanthemum yellows phytoplasmas (CYP) strain, and three others as non-vectors. Interactions between a labelled (recombinant) CYP Amp and insect proteins were analysed by far Western blots and affinity chromatography. Amp interacted specifically with a few proteins from vector species only. Among Amp-binding vector proteins, actin and both the α and β subunits of ATP synthase were identified by mass spectrometry and Western blots. Immunofluorescence confocal microscopy and Western blots of plasma membrane and mitochondrial fractions confirmed the localisation of ATP synthase, generally known as a mitochondrial protein, in plasma membranes of midgut and salivary gland cells in the vector Euscelidius variegatus. The vector-specific interaction between phytoplasma Amp and insect ATP synthase is demonstrated for the first time, and this work also supports the hypothesis that host actin is involved in the internalization and intracellular motility of phytoplasmas within their vectors. Phytoplasma Amp is hypothesized to play a crucial role in insect transmission specificity.
A novel, electrochemically synthesized, silver nanoparticles (AgNPs) formulation was evaluated
against
,
,
, and
strains from cystic fibrosis (CF) patients. AgNPs were particularly active against
and
...planktonic cells (median MIC: 1.06 and 2.12 μg/ml, respectively) by a rapid, bactericidal and concentration-dependent effect. AgNPs showed to be particularly effective against
and
biofilm causing a viability reduction ranging from 50% (1×MIC) to >99.9% (4×MIC). Electron microscopy showed that AgNPs deconstruct extracellular matrix of
biofilm, and accumulate at the cell surface causing cell death secondary to membrane damage. Compared to Tobramycin, AgNPs showed comparable, or even better, activity against planktonic and biofilm
cells. AgNPs at concentrations effective against
and
.
were not toxic to
larvae. Our silver-based formulation might be an alternative to antibiotics in CF patients. Further
and
studies are warranted to confirm this therapeutic potential.
Hypoxia, IL-1β production and oxidative stress are involved in islet graft dysfunction and destruction. However, the link between these events has not yet been determined in transplanted islets. The ...goal of this study was to determine whether NLRP3 inflammasome is responsible for IL-1β production and if it is activated by hypoxia-induced oxidative stress in transplanted islets. Rat islets were transplanted under the kidney capsule of immunodeficient mice. At different times post-transplantation, blood samples were collected and islet grafts harvested. Rat islets were also incubated in vitro either under normoxia or hypoxia for 24 h, in the absence or presence of inhibitors of NLRP3 inflammasome (CASP1 inhibitor) or oxidative stress (NAC). NLRP3, CASP1, IL1B, BBC3 pro-apoptotic and BCL2 anti-apoptotic genes in transplanted and in vitro incubated islets were then studied using real time PCR. IL-1β released in the blood and in the supernatant was quantified by ELISA. Cell death was analysed by propidium iodide and Annexin-V staining. NLRP3, CASP1 and BBC3 in transplanted rat islets and IL-1β in blood transiently increased during the first days after transplantation. In islets incubated under hypoxia, NRLP3, IL1B and CASP1 and IL-1β released in supernatant increased compared to islets incubated under normoxia. These effects were prevented by the inhibition of NLRP3 inflammasome by CASP1 or oxidative stress by NAC. However, these inhibitors did not prevent hypoxia-induced rat islet death. These data show that NLRP3 inflammasome in rat islets is transiently activated after their transplantation and induced through oxidative stress in vitro. However, NRLP3 inflammasome inhibition does not protect islet cells against hypoxia.
Numerous studies have investigated the role of the monounsaturated fatty acid and other dietary factors in the prevention of cognitive decline but the short-term effect of a low dose of extravirgin ...olive oil on cognitive performances in the elderly have not still been investigated. Our aim was to investigate whether the replacement of all vegetable oils with a lower amount of extravirgin olive oil, in the contest of a Mediterranean Diet, would improve cognitive performances, among elderly Italian individuals.
180 elderly individuals were randomly assigned to these treatment groups for 1 year: (1) MedDiet plus extravirgin OO, 20-30 g/day; (2) control MedDiet. The cognitive sub-test of ADAScale was used to detect cognitive decline progression over 12 months.
ADAS-cog score variation after 1 year, adjusted for food groups which were different between groups, was - 1.6 ± 0.4 and - 3.0 ± 0.4 in the MedDiet and MedDiet plus extravirgin OO groups, respectively (p = 0.024). Extravirgin OO intake was 30 g ± 12 and 26 g ± 6 in the MedDiet and MedDiet plus extravirgin OO groups, respectively (p = 0.044).
We demonstrated the higher short-term improvement of cognitive functions scores in individuals of the MedDiet plus low dose of extravirgin olive oil rather than MedDiet alone. Extravirgin olive oil is the best quality oil and may have a neuroprotective effect.
Phytoplasmas are uncultivated plant pathogens and cell wall-less bacteria and are transmitted from plant to plant by hemipteran insects. The phytoplasma's circulative propagative cycle in insects ...requires the crossing of the midgut and salivary glands, and primary adhesion to cells is an initial step toward the invasion process. The flavescence dorée (FD) phytoplasma possesses a set of variable membrane proteins (Vmps) exposed on its surface, and this pathogen is suspected to interact with insect cells. The results showed that VmpA is expressed by the flavescence dorée phytoplasma present in the midgut and salivary glands. Phytoplasmas cannot be cultivated at present, and no mutant can be produced to investigate the putative role of Vmps in the adhesion of phytoplasma to insect cells. To overcome this difficulty, we engineered the
mutant G/6, which lacks the ScARP adhesins, for VmpA expression and used VmpA-coated fluorescent beads to determine if VmpA acts as an adhesin in
adhesion assays and
ingestion assays. VmpA specifically interacted with
insect cells in culture and promoted the retention of VmpA-coated beads to the midgut of
In this latest case, VmpA-coated fluorescent beads were localized and embedded in the perimicrovillar membrane of the insect midgut. Thus, VmpA functions as an adhesin that could be essential in the colonization of the insect by the FD phytoplasmas.
Phytoplasmas infect a wide variety of plants, ranging from wild plants to cultivated species, and are transmitted by different leafhoppers, planthoppers, and psyllids. The specificity of the phytoplasma-insect vector interaction has a major impact on the phytoplasma plant host range. As entry into insect cells is an obligate process for phytoplasma transmission, the bacterial adhesion to insect cells is a key step. Thus, studying surface-exposed proteins of phytoplasma will help to identify the adhesins implicated in the specific recognition of insect vectors. In this study, it is shown that the membrane protein VmpA of the flavescence dorée (FD) phytoplasma acts as an adhesin that is able to interact with cells of
, the experimental vector of the FD phytoplasma.
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